Disappearance of GFP-Positive Hepatocytes Transplanted into the Liver of Syngeneic Wild-Type Rats Pretreated with Retrorsine

Background and Aim

Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP.

Methods

Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison.

Results

All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14.

Conclusion

GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.     

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The “high-expresser” line (GFP^high) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP^high-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The “low-expresser” line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.     

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.     

Development of new inbred transgenic strains of rats with LacZ or GFP

The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed most of their marker genes ubiquitously, compared to previous Tg rats. Immunological antigenicity against marker proteins was evaluated using conventional skin grafting, and results suggested lacZ-Tg-derived skin was much less immunogenic than that of GFP-Tg. However, GFP-positive cells from parental transgenic rats were still potential candidates for the study of cellular fate in immune privilege sites, such as the brain. Taking advantage of less immunogenic lacZ, we also examined the role of bone marrow-derived cells (BMDCs) in skin wound healing using an in vivo biological imaging system. Although transplantation of BMDCs enhanced wound healing at the injection site, BMDCs were detected only for a short time, suggesting a transient contribution of autologous BMDC-transplantation in wound healing. Our Tg-rat system may provide great benefits for the elucidation of the cellular process of regenerative medicine, including cell and tissue transplantation.     

New insight into mechanisms of allograft transplantation in the rat by differential display: macrophage scavenger receptor-A brings to light

BACKGROUND: Donor specific tolerance to heart allografts is induced in LEW.1A rat recipient by two donor LEW.1W blood transfusions prior engraftment. Although the tolerant allograft is infiltrated by leukocytes, graft infiltrating cells are only expressing low levels of the Th1- or Th2-related cytokines suggesting that induction of tolerance is an active phenomenon in which the mechanisms remain to be elucidated. MATERIALS AND METHODS: Differential display (DD) method was applied on RNAs extracted from graft infiltrating cells (GIC) derived from allografts either from rejecting untreated rats or donor-specific blood transfusion treated tolerant rats. Quantitative RT/PCR was performed to confirm mRNA expressions of the selected genes. RESULTS: Among the six differentially displayed DNAs (ddDNA) overexpressed in GIC from rejected allografts, the macrophage scavenger receptor-A (A:D13265) was identified; it exhibited a stricking induction of mRNA expression from day 1 to 7 after transplantation. Among the seven ddDNAs overexpressed in GIC from tolerant allografts, the 3-hydroxy-3-methyl glutaryl coenzyme-A reductase (A:M29249) and an "unknown gene" (ddDNA EC9) were identified and both were confirmed to be up-regulated by quantitative RT/PCR. CONCLUSIONS: The relevance of these genes in transplantation has not yet been reported and must therefore be elucidated; they represent possible targets for immunointervention. Nevertheless, our data demonstrate that the DD is a powerful tool to identify new genes involved in organ transplantation.     

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein-positive (GFP(+)) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP(+) hepatic cells. After reduced-size liver transplantation, GFP(+) cells were localized to the portal zone of the liver lobule. Interestingly, GFP(+) cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP(+) CD117(+) cells were identified in donor livers after 28 days. GFP(+) cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP(+) cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP(-) cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP(+) transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.     

Transfusion induces blood donor-specific suppressor cells

Transfusion with blood from the organ donor before transplantation can prolong the survival of renal allografts in the rat. To determine if the beneficial effect of preoperative blood transfusion was due to the generation of donor-specific suppressor cells, in vivo and in vitro adoptive transfer experiments were performed. Lymphoid cells were harvested from transfused and untreated rats. These cells were then either (1) transferred to lightly irradiated (200 R) syngeneic hosts which were subsequently challenged with a kidney allograft (in vivo assay) or (2) titrated as regulator cells into naive unidirectional MLC such that the regulator and responder populations were syngeneic. In the LEW-RT1 to DA-RT1av1 strain combination, the adoptive transfer of thoracic duct lymph (TDL) or lymph node (LN) cells (5 x 10(7) to 7.5 x 10(7) cells) from DA animals transfused with LEW blood, 7 days previously into syngeneic (DA), lightly irradiated (200 R) hosts resulted in the indefinite survival of LEW kidney allografts. The phenomenon was blood donor-specific and dose-dependent. In contrast the adoptive transfer of spleen cells (10(7) to 10(8] from blood transfused hosts 7 days after transfusion had no effect on renal allograft survival. In vitro the addition of LN or TDL regulator cells, harvested from DA rats transfused with LEW blood, to a unidirectional MLC (DA responders, LEW stimulators) resulted in a significant depression of the proliferative response when compared with the proliferation of these same cells without the addition of these regulator cells or with the addition of LN or TDL regulator cells from a DA rat transfused with third party (PVG-RT1c) blood. The depression of the proliferative response observed in vitro, was blood donor specific. When LN or TDL regulator cells from a DA rat transfused with PVG-RT1c blood were added to a unidirectional MLC between DA responders and PVG stimulators, a significant depression in the proliferative response was observed. These in vitro findings were confirmed in two other strain combinations (LEW-PVG, and DA-PVG). Thus a single blood transfusion results in the induction of donor-specific suppressor cells detectable both in vivo and in vitro 7 days after transfusion in some but not all lymphoid compartments.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats.

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric alpha-actin, cardiac alpha-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.     

Two phenotypically distinct populations of T cells have suppressor capabilities simultaneously in the maintenance phase of immunologic enhancement

The events leading to immunologic enhancement in LEW rats immunized actively with Brown Norway (BN) rat spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving (LEW X BN)F1 cardiac allografts, respectively, have been studied. Cellular suppressor mechanisms developing during the induction phase of this phenomenon have recently been shown to be mediated by W3/25+ T cells in an antigen-specific manner. The present study suggests that the late maintenance phase of immunologic enhancement is mediated in vivo by simultaneously present separate donor-specific T cell subpopulations of W3/25+ and OX8+ phenotypes. Splenocyte subsets from grafted recipients greater than 100 days after transplantation were adoptively transferred into unmodified syngeneic LEW rats that received a specific test allograft 24 hr later, or into B recipients bearing indefinitely surviving heart grafts. Test graft survival was prolonged significantly in the first group and not altered in the second. Indeed, nonoverlapping W3/25+ and OX8+ cell fractions were separately responsible for suppression. However, when suppressor activity was tested in vitro in a three-component coculture mixed lymphocyte reaction, no suppression by T cells was obtained; this lack of correlation between in vivo and in vitro results has also been noted by other investigators in different systems. Thus, in the maintenance phase of actively and passively induced immunologic enhancement, interplay between two phenotypically distinct T cells with suppressor characteristics, but not putative cell-surface blocking factors, seems to prevent development of an alloreactive response and mediate host unresponsiveness.     

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm³ after 3 days, 1.24 ± 0.76 mm³ after 7 days, 0.33 ± 0.03 mm³ after 14 days, and 0.09 ± 0.05 mm³ after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.     

转基因斑马鱼分析胰岛β-细胞发育情况

斑马鱼的个体小、高产和体外受精等特点使其已经迅速成为研究脊椎动物器官发育和人类疾病的模式生物之一。我们建立了一个转基因斑马鱼动物模型来研究胰岛β-细胞的发育。首先,构建了斑马鱼胰岛素（Insulin ,INS） 启动子与绿色荧光蛋白(GFP) 组成的表达载体, 命名为INS:GFP。其次,将质粒在斑马鱼1-细胞期注射到细胞质内。最后我们成功获得了生殖系稳定遗传胰岛素转基因斑马鱼,在成鱼和幼鱼期均可以通过GFP标记β-细胞。通过方便的荧光筛选,我们观察到胰岛在受精后18h开始形成,1－5d后由初始的脊索中线两侧向右迁移。从我们构建的胰岛素转基因斑马鱼,可以直观判断胰岛的发育情况,为研究胰岛的发育、损伤和再生提供了一个简便和直观的新型工具。     

W3/25+ T cells mediate the induction of immunologic unresponsiveness in enhanced rat recipients of cardiac allografts

Cellular suppressor mechanisms developing during the induction of immunologic enhancement were studied in LEW rats immunized actively with BN spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving a (LEW X BN)F1 cardiac allograft, respectively. With this regimen, graft survival is prolonged from 7.4 +/- 0.5 days in unmodified, acutely rejecting hosts to 25 +/- 12 days in enhanced recipients, with one-third of the grafts surviving indefinitely. To test for suppressor capacities, 60 X 10(6) splenic T helper/inducer (W3/25+) and T suppressor/cytotoxic (OX8+) cells were adoptively transferred 7 and 14 days after transplantation either into unmodified syngeneic LEW animals that received (LEW X BN)F1 test grafts 24 hr later or into T cell-deprived B rats with indefinitely functioning heart transplants that were reconstituted with sensitized lymph node cells (100 X 10(6). W3/25+ T cells harvested on days 7 and 14 from enhanced recipients prolonged test graft survival in unmodified hosts (13.1 +/- 2.3 and 13.3 +/- 1.3 days, respectively, p less than 0.001) and delayed rejection in reconstituted B rats from 6.7 +/- 0.5 to 18.2 +/- 6.5 and 23.3 +/- 5.8 days, respectively (p less than 0.001). OX8+ and B lymphocytes had no suppressor activity. However, enzymatic stripping of the surfaces of W3/25+ cells abrogated suppressor function. Similarly, after i.p. treatment with cyclophosphamide, W3/25+ T cells lost their suppressor properties. Lack of donor specific but not third party alloaggressiveness was demonstrated by the profoundly diminished ability of W3/25+ lymphocytes from enhanced hosts to recreate rejection in nonreconstituted B rats, even when exogenous interleukin 2 was administered. After pronase treatment, however, W3/25+ T cells were capable of inducing immunoresponsiveness at a tempo similar to naive T helper cells (31.5 +/- 12.5 vs 32.8 +/- 7.9). Thus, the present studies provide evidence for the development of a specific W3/25+ suppressor cell in the induction of active and passive enhancement. Coincident abrogation of specific T effector alloaggressiveness is apparently mediated by surface-blocking factors.     

绿色荧光转基因大鼠模型的建立

目的随着干细胞研究的推进,大鼠干细胞的研究日趋迫切。本研究旨在为活体荧光影像系统、干细胞归巢、细胞移植体内示踪研究,提供绿色荧光蛋白EGFP转基因大鼠模型。方法通过显微注射方式获得EGFP转基因大鼠,采用活体荧光影像系统、激光共聚焦显微镜,对EGFP转基因大鼠各个组织的荧光表达水平进行比较;采用流式细胞术检测转基因大鼠血液和骨髓细胞、骨髓干细胞的荧光标记率,筛选骨髓干细胞高效标记绿色荧光的转基因大鼠。结果建立了心脏、肝脏、肌肉、肺、胰腺、脑、膀胱、胃、肾脏、肠和脾脏组织中,系统性表达EGFP的SD-TgN（ACT-EGFP-1）ZLFILAS转基因大鼠;流式细胞术检测表明,该品系血液细胞绿色荧光标记率为94.4%,骨髓干细胞绿色荧光标记率为97.8%。结论建立了多组织系统性高表达绿色荧光,骨髓干细胞荧光标记率高达95%以上的转基因大鼠,为影像分析,造血干细胞的归巢等研究提供了大鼠模型。     

Rat chemokine CXCL11: Structure, tissue distribution, function and expression in cardiac transplantation models

CXCL11 is thought to play a critical role in allograft rejection. To clarify the role of CXCL11 in the rat transplantation model, we cloned CXCL11 cDNA from rat liver tissue and used it to study CXCL11 structure, function and expression. The rat CXCL11 gene encodes a protein of 100 amino acids and spans approximately a 2.8 kb DNA segment containing 4 exons in the protein coding region. Tissue distribution of rat CXCL11 was analyzed by quantitative RT-PCR and showed that rat CXCL11 mRNA is expressed in various tissues and, in particular, at high levels in the spleen and lymph nodes. COS-1 cells were transfected with a plasmid vector encoding rat CXCL11 and used to study CXCL11 effects on cell migration and internalization of CXCR3, the CXCL11 receptor. The recombinant CXCL11 showed chemotactic properties and induced CXCR3 internalization in CD4⁺ T cells. Expression of CXCL11 mRNA also was measured in rat acute (ACI to LEW) and chronic (LEW to F344) heart transplant rejection models. CXCL11 mRNA expression in allografts increased in both models, compared with controls, and was primarily observed in infiltrating macrophages and donor endothelial cells. These results indicate that, like the other CXCR3 chemokines, rat CXCL11 seems to have a role in the homing of CD4⁺ T cells in both acute and chronic rejection models of heart allotransplantation.     

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank’s Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.     

Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation.

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.     

Development and characterization of a new inbred transgenic rat strain expressing DsRed monomeric fluorescent protein

The inbred rat is a suitable model for studying human disease and because of its larger size is more amenable to complex surgical manipulation than the mouse. While the rodent fulfills many of the criteria for transplantation research, an important requirement is the ability to mark and track donors cells and assess organ viability. However, tracking ability is limited by the availability of transgenic (Tg) rats that express suitable luminescent or fluorescent proteins. Red fluorescent protein cloned from Discosoma coral (DsRed) has several advantages over other fluorescent proteins, including in vivo detection in the whole animal and ex vivo visualization in organs as there is no interference with autofluorescence. We generated and characterized a novel inbred Tg Lewis rat strain expressing DsRed monomeric (DsRed mono) fluorescent protein under the control of a ubiquitously expressed ROSA26 promoter. DsRed mono Tg rats ubiquitously expressed the marker gene as detected by RT-PCR but the protein was expressed at varying levels in different organs. Conventional skin grafting experiments showed acceptance of DsRed monomeric Tg rat skin on wild-type rats for more than 30 days. Cardiac transplantation of DsRed monomeric Tg rat hearts into wild-type recipients further showed graft acceptance and long-term organ viability (>6 months). The DsRed monomeric Tg rat provides marked cells and/or organs that can be followed for long periods without immune rejection and therefore is a suitable model to investigate cell tracking and organ transplantation.     

Analysis of green fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells.

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents--Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]--by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.     

Gene therapy strategies to facilitate organ transplantation.

Organ transplantation is now the definitive therapy for many forms of end-organ disease, but chronic allograft rejection, the side effects of chronic immunosuppressive therapy and the severe donor organ shortage continue to limit its success. Gene therapy has the potential to prevent graft rejection by manipulating the immune response in the microenvironment of the graft or by facilitating the induction of tolerance. Genetic manipulation of stem cells to create transgenic and/or knockout animals that could serve as organ or cell donors could be combined with gene therapy approaches to overcome the problem of limited allogeneic donor organ supply.